Introduction

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins responsible for signaling across cellular membranes. The initial signaling events are mediated by a wide variety of stimuli (including photons, Ca2+ ions, odorants, tasting molecules, amino acids, nucleotides, peptides, and proteins). The signal is transmitted to the cytoplasm, where downstream signaling is induced by receptor-mediated activation of G proteins. Based on pharmacological specificity and sequence conservation, GPCRs are divided into multiple classes.1 The three main classes are (A) receptors related to rhodopsins, (B) secretin receptors, and (C) the metabotropic neurotransmitter receptors. Class A, the largest, contains more than 1200 distinct members listed in the GPCR database1 and more than 7000 putative members in the Pfam family database.2

The GPCR family is characterized by a signature seven transmembrane (7TM) configuration that was first observed in bacteriorhodopsin by Henderson and Unwin3 and was subsequently proposed for rhodopsin, the mammalian dim-light receptor.4 High resolution crystal structures of bacteriorhodopsin confirmed the 7TM arrangement including extracellular (EC) loops and N-terminus, cytoplasmic (CP) loops and

FIGURE 11.1 Structure of rhodopsin. A: Crystal structure model. Helices are labeled I-VIII. Loops connecting the helices are labeled C-I, C-II, C-III and C-tail for the cytoplasmic side and N-tail; E-I, E-II, and E-III for the extracellular side. Glycosylation, retinal and disulfide bond are represented by sticks. B: Secondary structure model. Helices and beta sheets are marked by gray boxes and arrows, respectively. The disulfide bond between Cys187 and Cys110 is indicated by a dotted line and labeled with an arrow. All ten cysteines in rhodopsin are highlighted with circles.

FIGURE 11.1 Structure of rhodopsin. A: Crystal structure model. Helices are labeled I-VIII. Loops connecting the helices are labeled C-I, C-II, C-III and C-tail for the cytoplasmic side and N-tail; E-I, E-II, and E-III for the extracellular side. Glycosylation, retinal and disulfide bond are represented by sticks. B: Secondary structure model. Helices and beta sheets are marked by gray boxes and arrows, respectively. The disulfide bond between Cys187 and Cys110 is indicated by a dotted line and labeled with an arrow. All ten cysteines in rhodopsin are highlighted with circles.

C-terminus, binding of the all-trans retinal ligand, and detailed conformational changes upon photo-activation.5 Recent applications of atomic force microscopy to analysis of the conformational changes and folding dynamics of bacteriorhodopsin have also been reviewed elsewhere.6 Although bacteriorhodopsin has provided extensive information about the 7TM structure, it is not a true GPCR, lacking the ability to mediate the signal transduction events with downstream G protein signaling pathways.

The only GPCR for which information about the 7TM structure is directly available is bovine rhodopsin. First, a low resolution structure was derived by electron microscopy,7 followed by determination of the x-ray crystal structure for the inactive state8 (Figure 11.1A). Thus, rhodopsin, has proven to be a model par excellence for structural and functional analyses of GPCRs. The ligand in rhodopsin, 11-ds-retinal, is covalently attached to the protein via a protonated Schiff base to the e-amino group of Lys296 located in TM7 (Figure 11.1B). This gives rise to a characteristic absorption at 500 nm.

Activation of rhodopsin is achieved by light: capture of a photon by rhodopsin results in isomerization of 11-cis retinal to the all-trans form that triggers a series of transient changes in the protein accompanied by characteristic absorption changes: dark rhodopsin (500 nm) ^ batho (548 nm) ^ lumi (497 nm) ^ meta I (480 nm) ^ meta II (380 nm) ^ meta III (450 nm) ^ opsin + free retinal (380 nm).9 Only the meta II conformation can promote the activation of the G protein, the hallmark signaling event of the GPCR family.

This chapter will present the current state of knowledge of the structure and dynamics of rhodopsin in its dark conformation and in photo-intermediates, along with reviews of past and present state-of-the-art applied methodologies and in reference to other GPCRs in the ligand-free and ligand-bound states, where possible.